The long-term goal of this study is to identify the cause of Restless Legs Syndrome (RLS). Our working hypothesis for RLS is that the disorder results from brain iron insufficiency. This insufficiency could involve decreased iron uptake by cells, insufficient transport of iron to the brain, or insufficiency of iron acquisition by neurons. There are three fundamental questions that are raised in this proposal: 1) Is there a pathological change in the brain associated with RLS; 2) is there a functional deficit in the brain that could cause RLS; 3) what is the consequence of the functional deficit? These issues are addressed in this proposal using five aims: 1) we will use standard neuropathological evaluation of postmortem tissue to test the hypothesis that a neuroanatomical abnormality is causing the decreased iron uptake by the cells; 2) we will test the hypothesis that RLS results from insufficiency of iron acquisition. The consequences of iron insufficiency will focus on the integrity of the dopaminergic system. This evaluation includes analysis of Thy1, a protein involved in dopamine synaptic integrity, that we recently discovered is iron responsive. 3) We theorize that another possible cause of iron insufficiency in RLS brains is the dysfunctional delivery of iron to the brain. We propose to test the protein profile of the micro-vasculature, choroid plexus and ependymal cells in RLS. An abnormal protein profile at one of the interface sites between brain and plasma/CSF would suggest the site where regulation of brain iron uptake is altered; 4) to determine how the protein profile in the microvasculature is established and regulated we will use a whole animal model. The whole animal model will be used to test the hypothesis that the protein profile of the microvasculature is region specific and responds selectively to changes in systemic iron status; 5) to model the protein profile in the microvasculature to iron transcytosis and test the hypothesis that there are two mechanisms for iron delivery across the blood-brain barrier (BBB), we will use a cell culture model. At the conclusion of the proposed experiments, we will (i) have a profile of the RLS brain, (ii) have data that will either identify the cause of RLS or enable generation of hypotheses about the cause of RLS, (iii) develop novel research directions into the mechanisms of iron transport across the BBB and the regulation of those mechanisms.